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Journal Articles Journal of Biological Chemistry Year : 2005

Converting a beta-glycosidase into a beta-transglycosidase by directed evolution


Directed evolution was applied to the beta-glycosidase of Thermus thermophilus in order to increase its ability to synthesize oligosaccharide by transglycosylation. Wild-type enzyme was able to transfer the glycosyl residue with a yield of 50% by self-condensation and of about 8% by transglycosylation on disaccharides without nitrophenyl at their reducing end. By using a simple screening procedure, we could produce mutant enzymes possessing a high transferase activity. In one step of random mutagenesis and in vitro recombination, the hydrolysis of substrates and of transglycosylation products was considerably reduced. For certain mutants, synthesis by self-condensation of nitrophenyl glycosides became nearly quantitative, whereas synthesis by transglycosylation on maltose and on cellobiose could reach 60 and 75%, respectively. Because the most efficient mutations, F401S and N282T, were located just in front of the subsite (-1), molecular modeling techniques were used to explain their effects on the synthesis reaction; we can suggest that repositioning of the glycone in the (-1) subsite together with a better fit of the acceptor in the (-1) subsite might favor the attack of a glycosyl acceptor in the mutant at the expense of water. Thus these new transglycosidases constitute an interesting alternative for the synthesis of oligosaccharides by using stable and accessible donor substrates.
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hal-00385576 , version 1 (27-05-2021)


Attribution - CC BY 4.0



Hui Yong Feng, Jullien Drone, Lionel Hoffmann, Vinh Tran, Charles Tellier, et al.. Converting a beta-glycosidase into a beta-transglycosidase by directed evolution. Journal of Biological Chemistry, 2005, 280 (44), pp.37088-37097. ⟨10.1074/jbc.M502873200⟩. ⟨hal-00385576⟩
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